Mycobacterium avium Bacilli Grow Saprozoically in Coculture with Acanthamoeba polyphaga and Survive within Cyst Walls

Protozoans are gaining recognition as environmental hosts for a variety of waterborne pathogens. We compared the growth of Mycobacterium avium, a human pathogen associated with domestic water supplies, in coculture with the free-living amoeba Acanthamoeba polyphaga with the growth of M. avium when it was separated from amoebae by a 0.1-μm-pore-size polycarbonate membrane (in a parachamber). Although viable mycobacteria were observed within amoebal vacuoles, there was no significant difference between bacterial growth in coculture and bacterial growth in the parachamber. This suggests that M. avium is able to grow saprozoically on products secreted by the amoebae. In contrast, Legionella pneumophila, a well-studied intracellular parasite of amoebae, multiplied only in coculture. A comparison of amoebae infected with L. pneumophila and amoebae infected with M. avium by electron microscopy demonstrated that there were striking differences in the locations of the bacteria within amoebal cysts. While L. pneumophila resided within the cysts, M. avium was found within the outer walls of the double-walled cysts of A. polyphaga. These locations may provide a reservoir for the bacteria when environmental conditions become unfavorable.     

Survival of Environmental Mycobacteria in Acanthamoeba polyphaga

Free-living amoebae in water are hosts to many bacterial species living in such an environment. Such an association enables bacteria to select virulence factors and survive in adverse conditions. Waterborne mycobacteria (WBM) are important sources of community- and hospital-acquired outbreaks of nontuberculosis mycobacterial infections. However, the interactions between WBM and free-living amoebae in water have been demonstrated for only few Mycobacterium spp. We investigated the ability of a number (n = 26) of Mycobacterium spp. to survive in the trophozoites and cysts of Acanthamoeba polyphaga. All the species tested entered the trophozoites of A. polyphaga and survived at this location over a period of 5 days. Moreover, all Mycobacterium spp. survived inside cysts for a period of 15 days. Intracellular Mycobacterium spp. within amoeba cysts survived when exposed to free chlorine (15 mg/liter) for 24 h. These data document the interactions between free-living amoebae and the majority of waterborne Mycobacterium spp. Further studies are required to examine the effects of various germicidal agents on the survival of WBM in an aquatic environment.     

Acanthamoeba polyphaga-enhanced growth of Mycobacterium smegmatis

Background

Mycobacterium smegmatis is a rapidly-growing mycobacterium causing rare opportunistic infections in human patients. It is present in soil and water environments where free-living amoeba also reside, but data regarding M. smegmatis-amoeba relationships have been contradictory from mycobacteria destruction to mycobacteria survival.

Methodology/Principal Findings

Using optic and electron microscopy and culture-based microbial enumeration we investigated the ability of M. smegmatis mc² 155, M. smegmatis ATCC 19420^T and M. smegmatis ATCC 27204 organisms to survive into Acanthamoeba polyphaga trophozoites and cysts. We observed that M. smegmatis mycobacteria penetrated and survived in A. polyphaga trophozoites over five-day co-culture resulting in amoeba lysis and the release of viable M. smegmatis mycobacteria without amoebal cyst formation. We further observed that amoeba-co-culture, and lysed amoeba and supernatant and pellet, significantly increased five-day growth of the three tested M. smegmatis strains, including a four-fold increase in intra-amoebal growth.

Conclusions/Significance

Amoebal co-culture increases the growth of M. smegmatis resulting in amoeba killing by replicating M. smegmatis mycobacteria. This amoeba-M. smegmatis co-culture system illustrates an unusual paradigm in the mycobacteria-amoeba interactions as mycobacteria have been mainly regarded as amoeba-resistant organisms. Using these model organisms, this co-culture system could be used as a simple and rapid model to probe mycobacterial factors implicated in the intracellular growth of mycobacteria.     

Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis Survival within Amoebal Cysts

Simkania negevensis, a novel microorganism belonging to the family Simkaniaceae in the order Chlamydiales, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and S. negevensis has been associated with respiratory illness in humans. This study reports for the first time the ability of S. negevensis to survive and grow inside Acanthamoeba polyphaga in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of S. negevensis and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of S. negevensis in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of S. negevensis were observed within inclusion bodies inside A. polyphaga. When S. negevensis-infected A. polyphaga amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and S. negevensis; cysts in which S. negevensis cells were relegated to the space between cyst walls; and cysts containing S. negevensis, but apparently lacking amoebal cytoplasm. S. negevensis within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of S. negevensis needs to be investigated.     

Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms

Background

Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii.

Methodology/Principal Findings

Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10⁻⁶) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34×10⁴ CFU/mL) than M. tuberculosis (42×10¹ CFU/mL) and M. bovis (35×10¹ CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde.

Conclusions/Significance

These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.     

Interactions of Bacterial and Amoebal Populations in Soil Microcosms with Fluctuating Moisture Content

Sterilized soil samples (20 g of soil per 50-ml flask), amended with 600 μg of glucose-carbon and 60 μg of NH₄-N · g of dry soil⁻¹, were inoculated with bacteria (Pseudomonas paucimobilis) alone or with bacteria and amoebae (Acanthamoeba polyphaga). We used wet-dry treatments, which involved air drying the samples to a moisture content of approximately 2% and remoistening the samples three times during the 83-day experiment. Control treatments were kept moist. In the absence of amoebae, bacterial populations were reduced by the first drying to about 60% of the moist control populations, but the third drying had no such effect. With amoebae present, bacterial numbers were not significantly affected by the dryings. Amoebal grazing reduced bacterial populations to 20 to 25% of the ungrazed bacterial populations in both moisture treatments. Encystment was an efficient survival mechanism for amoebae subjected to wet-dry cycles. The amoebal population was entirely encysted in dry soil, but the total number of amoebae was not affected by the three dryings. Growth efficiencies for amoebae feeding on bacteria were 0.33 and 0.39 for wet-dry and constantly moist treatments, respectively, results that compared well with those previously reported for Acanthamoeba spp.     

Biodiversity of Amoebae and Amoeba-Resisting Bacteria in a Hospital Water Network

Free-living amoebae (FLA) are ubiquitous organisms that have been isolated from various domestic water systems, such as cooling towers and hospital water networks. In addition to their own pathogenicity, FLA can also act as Trojan horses and be naturally infected with amoeba-resisting bacteria (ARB) that may be involved in human infections, such as pneumonia. We investigated the biodiversity of bacteria and their amoebal hosts in a hospital water network. Using amoebal enrichment on nonnutrient agar, we isolated 15 protist strains from 200 (7.5%) samples. One thermotolerant Hartmannella vermiformis isolate harbored both Legionella pneumophila and Bradyrhizobium japonicum. By using amoebal coculture with axenic Acanthamoeba castellanii as the cellular background, we recovered at least one ARB from 45.5% of the samples. Four new ARB isolates were recovered by culture, and one of these isolates was widely present in the water network. Alphaproteobacteria (such as Rhodoplanes, Methylobacterium, Bradyrhizobium, Afipia, and Bosea) were recovered from 30.5% of the samples, mycobacteria (Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium xenopi) were recovered from 20.5% of the samples, and Gammaproteobacteria (Legionella) were recovered from 5.5% of the samples. No Chlamydia or Chlamydia-like organisms were recovered by amoebal coculture or detected by PCR. The observed strong association between the presence of amoebae and the presence of Legionella (P < 0.001) and mycobacteria (P = 0.009) further suggests that FLA are a reservoir for these ARB and underlines the importance of considering amoebae when water control measures are designed.     

Impact of Chlorine and Heat on the Survival of Hartmannella vermiformis and Subsequent Growth of Legionella pneumophila

Hartmannella vermiformis, a common amoebal inhabitant of potable-water systems, supports intracellular multiplication of Legionella pneumophila and is probably important in the transportation and amplification of legionellae within these systems. To provide a practical guide for decontamination of potable-water systems, we assessed the chlorine and heat resistance of H. vermiformis. H. vermiformis cysts and trophozoites were treated independently with chlorine at concentrations of 2.0 to 10.0 ppm for 30 min and then cocultured with L. pneumophila. Both cysts and trophozoites were sensitive to concentrations between 2.0 and 4.0 ppm and above (trophozoites somewhat more so than cysts), and 10.0 ppm was lethal to both forms. Hartmannellae treated with chlorine up to a concentration of 4.0 ppm supported the growth of legionellae. To determine whether heat would be an effective addendum to chlorine treatment of amoebae, hartmannellae were subjected to temperatures of 55 and 60°C for 30 min and alternatively to 50°C followed by treatment with chlorine at a concentration of 2 ppm. Fewer than 0.05% of the amoebae survived treatment at 55°C, and there were no survivors at 60°C. Pretreatment at 50°C appeared to make hartmannella cysts more susceptible to chlorine but did not further reduce the concentration of trophozoites.     

Crescent Bodies of Parachlamydia acanthamoeba and Its Life Cycle within Acanthamoeba polyphaga: an Electron Micrograph Study

Parachlamydiaceae are endosymbionts of free-living amoeba first identified in 1997. Two developmental stages, elementary and reticulate bodies, were observed; however, their localization and proportions according to culture condition and duration remain unknown. The life cycle of Parachlamydia acanthamoeba within Acanthamoeba polyphaga was studied by transmission electron microscopy of 8-, 36-, and 144-h coculture. Morphometry and quantification were performed using SAMBA software. The elementary body, the predominant stage within the amoebae, was located mainly within their vacuoles. The multiplication of Parachlamydia bacteria by binary fission of reticulate bodies was independently associated with culture in PYG broth (odds ratio [OR] = 4.4; 95% confidence interval [CI], 1.55 to 12.46) and with the presence of reticulate bodies within the amoebae (OR = 2.10; 95% CI, 1.53 to 2.89). A third developmental stage was observed, the crescent body. Its presence outside and inside the amoebae was associated mainly with prolonged incubation time (OR = 3.98; 95% CI, 1.49 to 10.68, and OR = 5.98; 95% CI, 1.75 to 20.4, respectively). Elementary and crescent bodies were released into the extracellular medium within vesicles or after amoebal lysis. For both, phagocytosis was their mode of entry. This electron micrograph study revealed another infective developmental stage, the crescent body, and provided quantitative analysis of the life cycle of P. acanthamoeba within A. polyphaga.     

Effects of Host Resistance on the Fecundity of Globodera rostochiensis

The fecundity of Globodera rostochiensis (R₁A) females that developed on resistant Rosa and susceptible Katahdin potato cultivars were compared. Cysts collected from each cultivar were bulked, separated into four sizes (> 500 μm, 355-500 μm, 250-355 μm, and < 250 μm), and crushed to determine fecundity as measured by viable egg content (VEC). Fewer and generally smaller cysts developed on Rosa than on Katahdin. Although cyst size significantly (P = 0.01) influenced VEC, cyst age (8 or 13 weeks) had no effect. Regardless of size, cysts produced on Rosa contained significantly fewer viable eggs than did cysts produced on Katahdin. The fecundity of progeny from cysts produced on Rosa was significantly reduced compared with that of progeny from cysts produced on Katahdin. After two generations on Katahdin, the VEC of cysts from a population originating from Rosa was significantly less than that of cysts from a population originating from Katahdin, indicating that in the presence of a pure population of G. rostochiensis R₁A, the females that develop on the resistant cultivar Rosa represent a diminished rather than a superior selected population.     

In Vitro Susceptibility of Atypical Mycobacteria To Rifampin

Atypical mycobacteria (209 strains) were examined for susceptibility to rifampin by the proportion method by using Middlebrook 7H-10 agar. All strains of Mycobacterium kansasii and tap-water scotochromogens were inhibited by 0.25 to 1 μg of the drug per ml. Seventy-six per cent of M. scrofulaceum and 61% of M. intracellulare strains were susceptible to 4 μg/ml or less; 5% of the former and 8% of the latter were resistant to 16 μg/ml. All strains of M. gastri and M. triviale and most strains of M. terrae were sensitive to 1 to 4 μg/ml. Two strains of M. borstelense were both inhibited by 8 μg/ml. Nearly all strains of M. fortuitum were resistant to the drug. The results of this study suggest that rifampin may be a valuable agent for the treatment of many atypical mycobacterial infections.     

Porins Increase Copper Susceptibility of Mycobacterium tuberculosis

Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 μM copper by 40% and completely suppressed growth at 15 μM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis.     

Schistosome sporocyst-killing Amoebae isolated from Biomphalaria glabrata.

Explants and swabs from the pericardium and mantle of three strains of Biomphalaria glabrata, two of them resistant to infection with Schistosoma mansoni, have yielded small amoebae, 3–5μm in diameter, in culture. These amoebae have been grown axenically through > 50 passages to date. The amoebae form cysts in dense cultures. When mixed with S. mansoni mother sporocysts in vitro, the amoebae adhere to and kill the trematodes within several hours. For 1–2 days thereafter, the amoebae proliferate rapidly at a generation time of about 5 hr, then return to normal growth. Sonically disrupted sporocysts also induce proliferation. Live sporocysts do not attract the amoebae or emit soluble substances which influence amoebal growth. Amoebae also adhered to and killed S. mansoni daughter sporocysts and cells derived from B. glabrata embryos; however, they did not harm S. mansoni cercariae or rediae of other trematode species. The proportion of mantle explants yielding amoebae was significantly higher (P<0.05) in one of the resistant snail strains than in the susceptible strain; however, whether amoebae contribute to snail resistance is unknown. Exposure of snails to S. mansoni miracidia did not influence the proportion of snails yielding amoebae.     

Occurrence of Free-Living Amoebae in Communities of Low and High Endemicity for Buruli Ulcer in Southern Benin

Buruli ulcer or Mycobacterium ulcerans disease occurs mainly in areas in proximity to standing or slowly running freshwater, habitats in which free-living amoebae occur. For this reason, a possible link between the habitat of M. ulcerans and free-living amoebae was investigated. Free-living amoebae and mycobacteria were isolated from water and biofilm specimens taken from protected and unprotected sources of water in villages known to have either high or low endemicity for Buruli ulcer in Benin. Amoebae were isolated from 78.8% of samples. A greater proportion of water bodies in areas of high endemicity had amoebae than in areas of low endemicity (83.3% versus 66.7%). Protected sources of water were significantly more likely to contain amoebae in areas of high endemicity than in areas of low endemicity (88.0% versus 11.1%). Several pathogenic free-living amoebae and mycobacteria were isolated. However, no M. ulcerans was isolated and no specimen was positive for IS2404 PCR. Our results show that the study area has a water hygiene problem, which is greater in areas of high Buruli ulcer endemicity than in areas of low endemicity. Our observations indicate that additional studies are required to explore the possible link between free-living amoebae and mycobacteria.     

Caulobacter crescentus as a Whole-Cell Uranium Biosensor

We engineered a strain of the bacterium Caulobacter crescentus to fluoresce in the presence of micromolar levels of uranium at ambient temperatures when it is exposed to a hand-held UV lamp. Previous microarray experiments revealed that several Caulobacter genes are significantly upregulated in response to uranium but not in response to other heavy metals. We designated one of these genes urcA (for uranium response in caulobacter). We constructed a reporter that utilizes the urcA promoter to produce a UV-excitable green fluorescent protein in the presence of the uranyl cation, a soluble form of uranium. This reporter is specific for uranium and has little cross specificity for nitrate (<400 μM), lead (<150 μM), cadmium (<48 μM), or chromium (<41.6 μM). The uranium reporter construct was effective for discriminating contaminated groundwater samples (4.2 μM uranium) from uncontaminated groundwater samples (<0.1 μM uranium) collected at the Oak Ridge Field Research Center. In contrast to other uranium detection methodologies, the Caulobacter reporter strain can provide on-demand usability in the field; it requires minimal sample processing and no equipment other than a hand-held UV lamp, and it may be sprayed directly on soil, groundwater, or industrial surfaces.     

Investigating the Role of Free-living Amoebae as a Reservoir for Mycobacterium ulcerans

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment.

Methodology/Principal Findings

We investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127).

Conclusion/Significance

This study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans.     

Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks,and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata

The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.     

Transformation of the Antibacterial Agent Norfloxacin by Environmental Mycobacteria

Because fluoroquinolone antimicrobial agents may be released into the environment, the potential for environmental bacteria to biotransform these drugs was investigated. Eight Mycobacterium sp. cultures in a sorbitol-yeast extract medium were dosed with 100 μg ml⁻¹ of norfloxacin and incubated for 7 days. The MICs of norfloxacin for these strains, tested by an agar dilution method, were 1.6 to 25 μg ml⁻¹. Cultures were extracted with ethyl acetate, and potential metabolites in the extracts were purified by high-performance liquid chromatography. The metabolites were identified using mass spectrometry and nuclear magnetic resonance spectroscopy. N-Acetylnorfloxacin (5 to 50% of the total absorbance at 280 nm) was produced by the eight Mycobacterium strains. N-Nitrosonorfloxacin (5 to 30% of the total absorbance) was also produced by Mycobacterium sp. strain PYR100 and Mycobacterium gilvum PYR-GCK. The MICs of N-nitrosonorfloxacin and N-acetylnorfloxacin were 2- to 38- and 4- to 1,000-fold higher, respectively, than those of norfloxacin for several different bacteria, including the two strains that produced both metabolites. Although N-nitrosonorfloxacin had less antibacterial activity, nitrosamines are potentially carcinogenic. The biotransformation of fluoroquinolones by mycobacteria may serve as a resistance mechanism.     

Acanthamoeba polyphaga Mimivirus Prevents Amoebal Encystment-Mediating Serine Proteinase Expression and Circumvents Cell Encystment

Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.